The long-term objective of this research is to elucidate the molecular mechanism by which the bacterial chaperone machine, composed of the 70-kDa molecular chaperone DnaK, the 41-kDa Co- chaperone DnaJ, and the 23-kDa nucleotide exchange factor GrpE, promotes protein folding. Several specific aims focus on understanding the chemistry of the interactions of DnaK with peptides, which serve to mimic unfolded substrate proteins. To be elucidated are how the total charge on a peptide affects its reactivity with both the low-affinity and high-affinity states of DnaK; how the alpha-helical lid affects the entry and exit of peptides into and out of the polypeptide-binding site of DnaK; and how a network of salt bridges between the beta-sandwich polypeptide-binding domain and the alpha-helical lid controls peptide binding to DnaK. Another specific aim is to dissect how the co-chaperones DnaJ and GrpB modulate the activity of DnaK. To be elucidated are the kinetics and mechanism of the DnaJ-assisted low- to-high affinity structural switch in DnaK; whether DnaJ lowers the activation energy barrier to peptide binding to the low-affinity state of DnaK; and the kinetics and mechanism of GrpE-assisted release of tightly bound polypeptides from the high-affinity state of DnaK. Variants of DnaK will be prepared using the tools of molecular biology. The specific aims will be achieved by conducting a variety of kinetic experiments both on the DnaK variants and the chaperone machine using stopped-flow fluorescence and surface plasmon resonance. There is an emerging awareness that as some cells age they may accumulate misfolded proteins, and that these misfolded proteins are linked to diseases such as amyloidosis, which can affect various organs. The expression of DnaK and DnaJ homologs by eukaryotic cells controls the amounts of unfolded proteins, reduces damage due to ischemia and even promotes the host attack against cancer cells; thus, elucidating the mechanism of the K/J/E chaperone machine may ultimately yield insights into the role of unfolded proteins in human diseases.